The thrust of this proposal is the examination of the mechanism for phosphoryl transfer in various enzyme catalyzed reactions. The experimental approach features an interplay between complimentary nonenzymatic and enzymatic investigations with the former serving as a guide for the mechanistic possibilities and the latter employing kinetic and stereochemical criteria to determine the locus of the rate determining step in the turnover sequence, the number and kinds of reaction intermediates, and ultimately to classify the reactions according to mechanistic types. Enzymatic reactions and enzymes to be investigated include: the hydrolyses of DNA catalyzed by various exonucleases including the 3' yields 5' exonuclease activity of E. coli polymerase I, exonuclease I, spleen phosphodiesterase, and restriction endonucleases; processive processes including the RNA polymerization catalyzed by polynucleotide phosphorylase and the 3' yields 5' degradation of single strand DNA by exonuclease I; the multiple activities of E. coli polymerase I and their integration into a unifying reaction sequence; the joining of DNA/RNA segments by ligases; and the proximity and orientations of ligands at the allosteric and active sites of fructose bisphosphatase (liver). These enzymes were selected owing to their importance in key metabolic pathways, such as gluconeogenesis and for the opportunity they afford for investigating important biochemical processes at the nucleic acid level namely polymerization, ligation, the fidelity of transcription and the phenomenon of processivity in addition to phosphoryl transfer. A description on the molecular level of how these reactions proceed provides insight into the operation of fundamental biochemical processes and holds promise for specific inhibitors designed on the basis of the mechanism of enzyme action that would be of diagnostic or therapeutic value.